Not applicable.
The invention provides isolated nucleic acid and amino acid sequences of Eag2, antibodies to Eag2, methods of detecting Eag2, and methods of screening for modulators of Eag2 potassium channels using biologically active Eag2. The invention further provides, in a computer system, a method of screening for mutations of human Eag2 genes as well as a method for identifying a three-dimensional structure of Eag2 polypeptide monomers.
Potassium channels are involved in a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, and regulation of renal electrolyte transport. Potassium channels are thus found in a wide variety of animal cells such as nervous, muscular, glandular, immune, reproductive, and epithelial tissue. These channels allow the flow of potassium in and/or out of the cell under certain conditions. For example, the outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels are made by alpha subunits that fall into 8 families, based on predicted structural and functional similarities (Wei et al., Neuropharmacology 35(7):805-829 (1997)). Three of these families (Kv, Eag-related, and KQT) share a common motif of six transmembrane domains and are primarily gated by voltage. Two other families, CNG and SK/IK, also contain this motif but are gated by cyclic nucleotides and calcium, respectively. The three other families of potassium channel alpha subunits have distinct patterns of transmembrane domains. Slo family potassium channels, or BK channels have seven transmembrane domains (Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25):14066-71 (1997)) and are gated by both voltage and calcium or pH (Schreiber et al., J. Biol. Chem. 273:3509-16 (1998)). Another family, the inward rectifier potassium channels (Kir), belong to a structural family containing 2 transmembrane domains (see, e.g., Lagrutta et al., Jpn. Heart. J. 37:651-660 1996)), and an eighth functionally diverse family (TP, or xe2x80x9ctwo-porexe2x80x9d) contains 2 tandem repeats of this inward rectifier motif.
Potassium channels are typically formed by four alpha subunits, and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). In addition, potassium channels such as those composed of Kv, KQT and Slo or BK alpha subunits have often been found to contain additional, structurally distinct auxiliary, or beta, subunits. These beta subunits do not form potassium channels themselves, but instead they act as auxiliary subunits to modify the functional properties of channels formed by alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify inactivation kinetics of the channel (Heinemann et al., J. Physiol. 493:625-633 (1996); Shi et al., Neuron 16(4):843-852 (1996)). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al., Nature 384:80-83 (1996)).
The Kv superfamily of voltage-gated potassium channels includes both heteromeric and homomeric channels that are typically composed of four subunits. Voltage-gated potassium channels have been found in a wide variety of tissues and cell types and are involved in processes such as neuronal integration, cardiac pacemaking, muscle contraction, hormone section, cell volume regulation, lymphocyte differentiation, and cell proliferation (see, e.g., Salinas et al., J. Biol. Chem. 39:24371-24379 (1997)).
A family of voltage-gated potassium genes, known as the xe2x80x9cEagxe2x80x9d or ether à go-go family, was identified on the basis of a Drosophila behavioral mutation with a leg-shaking phenotype (see, e.g., Warmke and Ganetzky, Proc. Nat""l Acad. Sci. USA 91:3438-3442 (1994)). Family members from Drosophila and vertebrates have been cloned and fall into three subfamilies. One such subfamily is called the Eag subfamily and is represented, e.g., by Drosophila Eag (Warmke et al., Science 252:1560-1562 (1991); Bruggemann et al., Nature 365:445-447 (1993)), and rat, mouse, human, and bovine Eags (Ludwig et al., EMBO J. 13:4451-4458 (1994); Robertson et al. Neuropharmacology 35:841-850 (1996); Occhiodoro et al., FEBS Letters 434:177-182 (1998); Shi et al., J. Physiol. 115.3:675-682 (1998); Frings et al., J. Gen Physiol. 111:583-599 (1998)). A second subfamily, the Erg or xe2x80x9cEag-related genexe2x80x9d family is represented, e.g., by human erg (Shi et al., J. Neurosci. 17:9423-9432 (1997)). Finally, a third subfamily, the Elk or xe2x80x9cEag-like K+ genexe2x80x9d is represented, e.g., by Drosophila Elk (Warmke et al., Proc. Natl. Acad. Sci. 91:3438-3442 (1994)).
The present invention thus provides for the first time Eag2, a polypeptide monomer that is an alpha subunit of an voltage-gated potassium channel. Eag2 has not been previously cloned or identified, and the present invention provides the nucleotide and amino acid sequence of human Eag2.
In one aspect, the present invention provides an isolated nucleic acid encoding an alpha subunit of a potassium channel, wherein the subunit: (i) forms, with at least one additional Eag family alpha subunit, a potassium channel having the characteristic of voltage sensitivity; and (ii) comprises an amino acid sequence that has greater than about 70% identity to amino acids 720-988 of a human Eag2 amino acid sequence or comprises an amino acid sequence that has greater than about 85% identity to the amino acid sequence of SEQ ID NO:2.
In another aspect, the present invention provides an isolated nucleic acid that selectively hybridizes under moderately stringent hybridization conditions to a nucleotide sequence of SEQ ID NO:1.
In another aspect, the present invention provides an isolated nucleic acid that selectively hybridizes under stringent conditions to a nucleotide sequence of SEQ ID NO:1 or to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:2.
In another aspect, the present invention provides a method of detecting a nucleic acid, by contacting the nucleic acid with a nucleic acid of the invention.
In another aspect, the present invention provides an isolated alpha subunit of a potassium channel, wherein the subunit: (i) forms, with at least one additional Eag family alpha subunit, a potassium channel having the characteristic of voltage sensitivity; (ii) comprises an amino acid sequence that has greater than about 70% identity to amino acids 720-988 of a human Eag2 amino acid sequence or comprises an amino acid sequence that has greater than about 85% identity to the amino acid sequence of SEQ ID NO:2.
In one embodiment, the polypeptide specifically binds to polyclonal antibodies generated against SEQ ID NO:2.
In one embodiment, the nucleic acid encodes human Eag2. In another embodiment, the nucleic acid encodes SEQ ID NO:2. In another embodiment, the nucleic acid has the nucleotide sequence of SEQ ID NO:1. In another embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as primers selected from the group consisting of:
ATGCCGGGGGGCAAGAGAGGGCTG (SEQ ID NO:3);
CTGACCCTAAGCTCATAAGGATGAAC (SEQ ID NO:4);
CCACCTCATCATCCTGGATGACTTCC (SEQ ID NO:5),
TTAAAAGTGGATTTCATCTTTGTCAGATTCAGG (SEQ ID NO:6);
GGGGACCTCATTTACCATGCTGGAG (SEQ ID NO:7); and
GATTCCCTCATCCACATTTTCAAAGGC (SEQ ID NO:8).
In another embodiment, the polypeptide monomer has a molecular weight of between about 109 kD and about 119 kD. In another embodiment, the polypeptide monomer has the sequence of SEQ ID NO:2.
In another embodiment, the polypeptide monomer comprises an alpha subunit of a heteromeric or homomeric potassium channel.
In another aspect, the present invention provides an expression vector comprising the isolated nucleic acid and a host cell transfected with such an expression vector.
In another aspect, the present invention provides an antibody that selectively binds to the isolated polypeptide monomer.
In another aspect, the present invention provides a method for identifying a compound that increases or decreases ion flux through a potassium channel, the method comprising the steps of: (i) contacting the compound with an alpha subunit of a potassium channel, wherein the subunit: (a) forms, with at least one additional Eag family alpha subunit, a potassium channel having a characteristic of voltage sensitivity; and (b) comprises an amino acid sequence that has greater than about 70% to amino acids 720-988 of a human Eag2 amino acid sequence or comprises an amino acid sequence that has greater than about 85% identity to the amino acid sequence of SEQ ID NO:2; and (ii) determining the functional effect of the compound upon the potassium channel.
In one embodiment, the functional effect is determined by measuring changes in ion flux, current, voltage, or ion concentration. In another embodiment, the polypeptide monomer is recombinant.
In one embodiment, the functional effect is a physical effect. In another embodiment, the functional effect is a chemical effect. In another embodiment, the functional effect is determined by measuring ligand binding to the channel.
In one embodiment, the polypeptide is expressed in a cell or cell membrane, e.g., a eukaryotic cell or a human cell. In another embodiment, the polypeptide is attached to a solid support.
In another aspect, the present invention provides a method of detecting the presence of human Eag2 in a biological sample, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a human Eag2-specific reagent that selectively associates with human Eag2; and, (iii) detecting the level of human Eag2-specific reagent that selectively associates with the sample.
In one embodiment, the human Eag 2-specific reagent is selected from the group consisting of: Eag 2-specific antibodies, Eag 2-specific oligonucleotide primers, and Eag 2-specific nucleic acid probes.
In another aspect, the present invention provides, in a computer system, a method of screening for mutations of a human Eag2 gene, the method comprising the steps of: (i) entering into the system at least about 50 nucleotides of a first nucleic acid sequence encoding a human Eag2 gene having a nucleotide sequence of SEQ ID NO:1, and conservatively modified variants thereof; (ii) comparing the first nucleic acid sequence with a second nucleic acid sequence having substantial identity to the first nucleic acid sequence; and (iii) identifying nucleotide differences between the first and second nucleic acid sequences.
In one embodiment, the second nucleic acid sequence is associated with a disease state. In another embodiment, the step of entering comprises entering into the system a nucleotide sequence corresponding to amino acids 720-988 of a human Eag2 gene encoding polypeptide having an amino acid sequence of SEQ ID NO:2.
In another aspect, the present invention provides a method for identifying a compound that increases or decreases ion flux through a potassium channel comprising an Eag2 polypeptide, the method comprising the steps of: (i) entering into a computer system an amino acid sequence of at least 50 amino acids of an Eag2 polypeptide or at least 150 nucleotides of a nucleic acid encoding the Eag2 polypeptide, the Eag2 polypeptide comprising a subsequence having at least 70% amino acid sequence to amino acids 720 to 988 of SEQ ID NO:2 or comprises an amino acid sequence that has greater than about 85% identity to the amino acid sequence of SEQ ID NO:2; (ii) generating a three-dimensional structure of the polypeptide encoded by the amino acid sequence; (iii) generating a three-dimensional structure of the potassium channel comprising the Eag2 polypeptide; (iv) generating a three-dimensional structure of the compound; and (v) comparing the three-dimensional structures of the polypeptide and the compound to determine whether or not the compound binds to the polypeptide.
In another aspect, the present invention provides a method of modulating ion flux through an Eag potassium channel to treat disease in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified using the methods of the invention.